Protein purification 19-23

What is an assay?

A way to measure something

What is Vo?

change in absorbance/change in time

What do all proteins have in common?

1. Peptide Bonds

2. Hydrophobic R-groups

Is the Bradford assay sensitive? What does it do?

-Yes it is very sensitive and also easy to do

-It uses coomassie blue to bind to hydrophob

-Once the dye binds to proteins it changes color from red to blue

-blue means A595nm

-Measures all proteins, not your protein specifically

What does specific activity tell you?

-how active your enzyme is

How do you calc specific activity?

I.U/mg of protein

Ex: 3500 I.U/10,000mg = 0.35 I.U/mg

What does fold purification tell you?

how much purer your enzyme sample has become after a purification step, compared to the starting crude extract

How do you calculate fold purification?

specific activity of current fraction/specific activity of crude

7.2 I.U./mg / 0.35 I.U/mg = 20.57 = 20x more pure than crude

What does % yield enzyme activity tell you?

tells you how much of the enzyme's original activity you kept after a purification step, compared to the crude extract

How do you calculate % yield enzyme activity?

I.U in current step/I.U in crude x100

(2500 I.U)/(3500 I.U) x 100 = 71%

How do you calculate % yield protein

(mg protein in current step/mg protein in crude) x 100

How do we know if a protein is pure?

1. No further increase in specific activity

2. Can look at the # of bands of a gel

How does a Native PAGE gel separate proteins?

1. size (larger = move slower)

2. Charge to mass ratio (more - = migrate faster)

How does an SDS-PAGE gel separate proteins?

By molecular weight

-SDS denatures proteins and gives them a uniform negative charge.

-It destroys hydrophobic effect

What are three ways to break open cell?

1. Waring blender

2. Sonicator

3. N2

What does a waring blender do?

It shears cells, animal and plant tissue but not microbes

-In blender you add cold buffer and a bunch of tissue and blend up

-You need to keep cold because most proteins last longer if kept cold

What does a sonicator do?

It uses sound waves and this excites solvent water molecules to move away from the cell

-Energy coming out of the probe will heat the solution

What does liquid nitrogen do?

Put tissue and N2 and water molecules expand (cold makes expansion) and rip everything apart

-Then take buffer and put on top (provide enviro for assays)

Describe an experiment to take spinach tissue and turn it into a crude extract

1. Take you spinach tissue and 20L of buffer and put in a waring blender to break apart the cells

2. Then centrifuge that mixture for 30 minutes

3. You will have a fat pad of lipids at the top, supernatant, and a pellet (holds things that dont want to go in solution)

4. collect the supernatant and now you have your crude extract

Why is the crude extract a problem aka what must we reduce and remove

1. need to reduce the volume (proteins last longer in more conc solution)

2. need to remove nucleic acid at CHO polymers

How can we "fix" the crude extract? What can we use?

We can use ammonium sulfate. It will make 2 cuts

1. the first cut is to remove nucleic acids and CHO polymers

2. the second cut is to reduce the volume and be able to store the enzyme

Gives 2 fold purification

How does ammonium sulfate "cut"?

-Proteins dissolve in water because water molecules form hydration shells around them.

Ammonium and sulfate strongly bind water so this removes water molecules from proteins. With less water the proteins will bind to each other and then fall out as precipitate

How can we do an experiment with ammonium sulfate in the goal of running an assay?

1. take crude extract and put in grams ammonium sulfate and put in beaker and mix.

2. centrifuge and you will get 20% A.S supernatant and a white pellet

3. Take 20% supernatant and more grams A.S and put in different breaker and centrifuge/mix

4. You will get 60% ammonium sulfate pellet (where target enzyme is) and you can store in freezer and when you are ready to use you can dissolve in water

5. Then we can do an enzyme Z assay (tells you how much enzyme activity you have recovered) and a bradford protein assay (measures protein conc, helps estimate purity)

What does a sizing column do?

Why is it a good technique?

It separates by molecular weight

Can you large, medium or small pore depending on the size of your protein

It is a good technique because it is gentle and you dont lose enzyme activity

Do you want the bead for a sizing column to be polar or nonpolar?

You want it to be polar because you don't want your enzyme to open up.

What happens if you do a sizing colomn and have no activity?

This means you protein might need a coenzyme so you can check a later fraction and add the coenzyme to the protein peak and see if it bring it back to life.

Does a sizing column disturb protein structure?

No, 1, 2, 3, 4 is still intact

What fold purification does a sizing column give?

10x

What does the area under a peak in a elution profile mean?

The area under a peak is proportional to how much you have

What happens if you only get one peak in your elution profile?

This means that all the proteins have the same molecular weight

How does an ion exchange column separate proteins?

It separates protein based on charge not size

-so you want large pores

Why is the resin in an ion exchange column polar?

It is polar because it is made of sugar

What is DEAE-Sephadex?

It is an anion exchanger.

It has a nitrogen that has a pka of about 9 and if pH is lower than 9 it will grab. proton and be positively charged and be able to exchange anions

What is CM-Sephadex?

It is a cation exchanger.

-O-CH2-COOH . the H has a pka of 4 so if pH is more than 4 it will be negatively charged and exchange cations

How do you get your protein off the resin after it binds?

Ion exchange column

1. change pH, it will alter the charge on the protein/resin. no longer binding and can elute

2. Put in more salt (NaCl). Salt ions "shield" the resin's charge → proteins no longer stick → they elute.

can do a pH gradient also a salt gradient to help proteins elute more slowly

What does affinity chromatography do?

purifies proteins based on specific biological interactions

How does affinity chromatography work?

There is a polar brad that has carbon arms sticking out of it with ligands (S, P, I Coenz, Cofact, etc) attached to it.

Only biologically active enzymes have the right active site to bind the ligand (Inactive or misfolded enzymes won't bind)

How do you determine purity after you do affinity chromatography?

You can do a bradford assay to determine # of proteins.

Load your sample → the target enzyme binds the ligand

Wash → removes junk proteins

Elute:

Using a buffer with ligand or high salt (salt washes off proteins we are not interested in)

Only your target enzyme comes off

You see a small blue peak (little protein)

BUT a strong red peak (high enzyme activity)

This shows high purity.

Why might affinity chromatography fail?

1. The ligand might be in the wrong orientation and might not fit perfectly with enzyme active site

2. Binding order matters

Example:

The enzyme needs two cofactors S and S* in a specific order. If the column presents them incorrectly, binding won't happen.

How does an IEF gel separate proteins?

Separates by pI (pH where protein has zero charge)

How does an IEF gel work?

1. You cast a gel containing many ampholytes. Ampholytes = small molecules that each have their own pI.

2. Apply an electric field →Ampholytes arrange themselves into a stable pH gradient from pH ~3 to ~10.

3. Add your proteins:

A protein that is positive at a given pH moves toward the negative electrode

A protein that is negative moves toward the positive electrode

4. When it reaches the pH that matches its pI, it stops, because net charge = 0.

High pI protein → ends up at the high pH side

Low pI = end up at the low pH side

What is the watman paper near the cathode side soaked with?

What is the anode side soaked wit?

1M HCl to allow for a resivour of H+

1M NaOH to allow for OH-

How would you then test enzyme purity after you run a IEF gel?

You take 1 lane from your preparitive IEF gel and put in in a tube with deionized water (this lets the proteins diffuse out of the gel into solution)

Then you do a bradford assay and get different protein peaks. You can plot where you get most enzyme activity (enzyme activity assay) and scoop this area out and put it on a different IEF gel. Say for example you saw a large peak from pH 6-8 you would buy amphalites at from pH 6-8 (creates the pH gradient)

Narrow pH gels = good for purifying it

Are analytical PAGE gels used for purification?

No, instead it is a way to visually check if your protein is pure

Why would you use polyacrylamide for a gel instead of agrose?

1. It is stronger than agrose

2. You get to control pore size

The gel allows for reduced diffusion and the moleculer sieve allows separation by molecular weight.

Does a 4% weight:volume ration give large or small pores?

Large pores

Does a 10% w/v ration give large of small pores

smaller pores

What does ammonium persulfate do to a polyacrylamide gel?

APS initiates polymerization of acrylamide, without it the gel can never form

It creates a free radical chain reaction

What does tetramethylethylenediamine (TEMED) do regarding a polyacrylamide gel?

It stabilizes the free radicals

What is analytical IEF gel?

It is horizontal and you use a pH gradient made by amphilites

You can have a mixture of proteins applied to the middle and they will separate based on their pI

High pI = go to anode side with high pH

low pI = go to cathode side with low pH

What does a native PAGE gel separate by?

charge and mass

What does a SDS page gel separate by?

size only, it gives all proteins same charge to mass ratio

Denatures proteins

Destroys hydrophobic effect

Gives all proteins same charge/mass ratio

Native PAGE gel

1. What is the purpose of a stacking gel?

2. What part of the gel do the proteins separate?

3. What are the 4 things you will pipet in the well?

4. What is the purpose of glycerol?

5. What causes uneveness at the bottom of the gel

6. Do you want large or small pores?

1. To compress the sample into a thin line

2. In the resolving gel, middle part

3. The protein, pH 6.8 (pH of stacking gel), glycerol, bromophenol blue

4. It increases the density of the solution so you don't contaminate the other wells

5. Salt ions in each sample

6. Large pores because you want all proteins to be able to stack

Can you see subunits on a native page gel?

no

Why do you stain the native page gel with comassie blue?

What does destaining the solution do?

Because comassie blue will bind to hydrophobic groups and will denature the proteins in place

Stain not bound to proteins will diffuse out

How do you find enzyme activity on a native page gel

Take a lane and take a small slice and assay for enzyme activity

Why run a native page gel?

1. estimate purity

the protein remains folded and active. If you see one band that means it is fairly pure. Helps you answer is your last purification step worked

2. determine native MW. You are able to estimate whole complex since the 4 structure is held together

You run a series of native page gels at different pore sizes and see how much they move bc migration depends on charge, size, and shape

3. helps you determine next purification step.

If proteins separate well by charge → ion exchange is a good next step

If they don't separate by charge → try size-based or affinity methods

What happens if you change pH of the resolving gel?

You change the charge because charge depends on pH and you change the charge to mass ratio

What does SDS do?

binds hydrophobic regions

unfolds protein

coats backbone with negative charge

have only primary structure left

How would you break apart a tetramer of hemoglobin

Take Hb and treat with xs SDS, xs mercaptoethanol (break disulfide bonds), and head to speed up rxn and break weak bonds

What do you put in when you want to run an SDS page gel?

What do you have where the buffer would be?

What do you do after the gel has been run?

You have a stacking gel and resolving gel just like native PAGE gel

You put in your protein, SDS, mercapotoethanol, glycerol and bromophenol blue into wells.

You have SDs where buffer would be. You have SDS all throughout gel

After the gel is run you stain and then destain

What does SDS page gel separate by?

Molecular weight

Why would you run an SDS page gel?

1. estimate purity (get 20 bands = not pure)

2. determine subunit MW by putting standards in 1 lane

3. look at proteins buried in membrane (cannot put membrane on gel so treat with SDS and get out of membrane)

How can you get # of subunits from data from native PAGE gel and SDS page gel?

say in a native page gel you get Hb = 68,000 MW

in sds page gel you get Hb = 17,000 MW

This tells you that you have 4 subunits. If you get 68,000 in SDS page gel that means you have no subunits

But it does not tell you that you have 2 alpha and 2 beta